Shafqat F. Rehmani

Phylogenetic and biological characterization of Newcastle disease virus isolates from Pakistan.

ABSTRACT Eight Newcastle disease virus isolates from Pakistan were sequenced and characterized. A PCR matrix gene assay, designed to detect all avian paramyxovirus 1, did not detect four of the isolates. A new matrix gene test that detected all isolates was developed. Phylogenetic analysis and pathotyping confirmed that virulent viruses of different genotypes are circulating in Pakistan.

Sequence and phylogenetic analysis of H7N3 avian influenza viruses isolated from poultry in Pakistan 1995-2004

BackgroundAvian influenza virus (AIV) infections have caused heavy economic losses to the poultry industry in Pakistan as well as numerous other regions worldwide. The first introduction of H7N3 AIV to Pakistan occurred during 1995, since then H7N3, H9N2 and H5N1 AIVs have each been sporadically isolated. This report evaluates the genetic origin of the H7N3 viruses from Pakistan collected 1995-2004 and how they disseminated within the country. To accomplish this we produced whole genome sequences for 6 H7N3 viruses and data for the HA and NA genes of an additional 7 isolates. All available sequence from H7N3 AIV from Pakistan was included in the analysis.ResultsPhylogenetic analysis revealed that there were two introductions of H7 into Pakistan and one N3 introduction. Only one of the H7 introductions appears to have become established in poultry in Pakistan, while the other was isolated from two separate outbreaks 6 years apart. The data also shows that reassortment has occurred between H7N3 and H9N2 viruses in the field, likely during co-infection of poultry. Also, with the exception of these few reassortant isolates, all 8 genes in the predominant H7N3 virus lineage have evolved to be phylogenetically distinct.ConclusionsAlthough rigorous control measures have been implemented in commercial poultry in Pakistan, AIV is sporadically transmitted to poultry and among the different poultry industry compartments (broilers, broiler breeders, table egg layers). Since there is one primary H7 lineage which persists and that has reassorted with the H9N2 AIV in poultry, it suggests that there is a reservoir with some link commercial poultry. On a general level, this offers insight into the molecular ecology of AIV in poultry where the virus has persisted despite vaccination and biosecurity. This data also illustrates the importance of sustained surveillance for AIVs in poultry.

Newcastle disease vaccination: A comparison of vaccines and routes of administration in Pakistan

Abstract Twelve-day-old chickens were vaccinated once with different Newcastle disease (ND) vaccines ( F, La Sota and Mukteswar) by two different routes (intraocular and drinking water). Chickens from a seventh group were uninoculated controls. At weekly intervals for 7 weeks after vaccination, 20 chickens from each vaccinated group and 20 chickens from the control group were examined for the production of haemagglutination-inhibition (HI) antibodies and for protection as assessed after challenge with velogenic, viscerotropic ND virus. La Sota ND vaccine used intraocularly ranked the best and Mukteswar vaccine by the drinking water route the worst for their HI antibody titres prior to challenge. Differences between the treatments in protection were examined. For all three vaccines intraocular vaccine produced higher protection than drinking water vaccine. An inverse relationship between prechallenge and postchallenge HI titres was also recorded.

The influence of adjuvants on oral vaccination of chickens against Newcastle disease

Living V4 strain Newcastle disease vaccine was given to chickens orally. The inclusion of DEAE-dextran, Quil-A or TiterMax in the vaccine, or delivering the vaccine as Iscoms, did not enhance the serological response. The immediate serological response to living V4 vaccine was enhanced in the presence of Avridine. Chickens produced a low serological response to oral administration of inactivated V4 vaccine. This response was not enhanced in the presence of Avridine.

Lactose pellets — a new approach to oral vaccination of village chickens against Newcastle disease

Newcastle disease in village chickens in developing countries can now be controlled with vaccines containing thermostable, avirulent V4 virus delivered on food. Logistical problems arise because 7 to 10 g of food vaccine must be allowed for each chicken. Lactose-based pellets have been prepared that contain an immunizing dose of V4 virus in a single pellet, even after long periods of storage. Protective levels of antibody were generated in chickens fed individual pellets, or in groups of chickens fed vaccine pellets mixed with normal food. Chickens receiving vaccine pellets developed a level of protection against challenge with virulent Newcastle disease virus similar to that achieved with vaccine added to food. This process when refined will allow the preparation of vaccine in regional laboratories and delivery without refrigeration to villages.

Receptors for the V4 strain of Newcastle disease virus in the digestive tract of chickens

Enterocytes were detached from various parts of the digestive tract of chickens by treatment with DTT or with hyaluronidase. Isolated enterocytes were exposed to suspensions of the V4 strain of Newcastle disease virus (NDV). Removal of virus from the supernatant fluid was taken as evidence of binding of virus to enterocytes and residual virus was measured both by infectivity assay and by ELISA. Enterocytes from duodenum, jejunum, ileum, caecum, and rectum bound the virus; enterocytes from oesophagus, crop and proventriculus did not.

Characterization of H5N1 highly pathogenic avian influenza viruses isolated from poultry in Pakistan 2006–2008

Nine avian influenza viruses (AIV), H5N1 subtype, were isolated from dead poultry in the Karachi region of Pakistan from 2006 to 2008. The intravenous pathogenicity indices and HA protein cleavage sites of all nine viruses were consistent with highly pathogenic AIV. Based on phylogenetic analysis of the HA genes, these isolates belong to clade 2.2 and both the HA and NA are closely related to each other (nucleotide identities above 99.0%) and to other Middle Eastern H5N1 AIV isolates (nucleotide identities above 98.0%). The phylogenetic data suggest that the virus in both epornitics of H5N1 HPAIV in commercial poultry in the Karachi region of Pakistan between 2006 and 2008 were from a very closely related source, however, there is inadequate epidemiological data to determine what the reservoir was for the virus between the 2006 and 2007 outbreaks other than that there was a single introduction into the region.

Passive immunity in chicks from a laying flock vaccinated with the Mukteswar strain of Newcastle disease virus

Pullets about to enter a laying flock were revaccinated by intramuscular injection of live Mukteswar strain Newcastle disease virus, a procedure commonly used in Pakistan. Chicks hatched from eggs produced 24 weeks later were assessed for passively acquired immunity. Levels of haemagglutination-inhibition (HI) antibody and resistance to challenge with velogenic Newcastle disease virus were determined on groups of 50 chicks at 1, 4, 7, 10, 13, 16, 19, 22 and 25 days of age. Levels of maternally derived antibody declined from a geometric mean titre of 40 (25.3) on the day of hatching to less than two (20.3) at 25 days of age, with a decline of 1log2 in about 5 days. Mortalities after challenge varied from 18% at 1 day of age to 90% at 25 days of age. Survival was significantly related to the level of antibody at the time of challenge. None of 111 chickens with an antibody titre of 40 (25.3) or more succumbed to challenge. Mortality rates varied from 78.6% in chicks with no detectable antibody at challenge to 27.6% in chicks with a titre of 20 (24.3). Levels of HI antibody and neutralizing antibody were strongly correlated.

The contribution of lectins to the interaction between oral Newcastle disease vaccine and grains

Successful oral vaccination of chickens with Newcastle disease (ND) depends on the survival of vaccine virus on the grains that are used as carriers. Some interactions between grains and the V4 strain of ND virus (NDV) were studied. Crude saline washings were prepared from several grains - rice (unhusked, brown, white and boiled white), sorghum, millet, wheat, maize and barley - and tested for lectin activity, as indicated by agglutination of chicken erythrocytes. Only washings from unhusked rice, sorghum and millet failed to haemagglutinate. None of the crude washings antagonised the haemagglutinating activity of NDV, and the washing from white rice produced an 8-fold enhancement. The presence of lectins in the washings from rice, wheat and barley was confirmed by purifying a substance with N-acetyl-D-glucosamine specificity. Only the crude extract from white rice had any profound effect on infectivity, reducing the infectivity titre by 99.99%. It is not known if the viricidal substance is identical with the lectin. Of 9 commercial lectins tested, only ConA bound the V4 virus.

Molecular Characterization of Pakistani Field Isolates of Infectious Bursal Disease Virus

Abstract The reverse transcriptase-polymerase chain reaction followed by restriction fragment length polymorphism (RT-PCR/RFLP) technique was used to identify and characterize Pakistani field isolates of infectious bursal disease virus (IBDV). These isolates have caused heavy losses to the poultry industry (mortality up to 60%) during the period between 1999 and 2005. Ten samples (five local isolates and five commercial vaccines) were examined for IBDV. Nine samples were positive for IBDV as evidenced by the amplification of the 743-bp region of the VP2 gene by RT-PCR. The RT-PCR products were subjected to restriction enzyme digestion with BstNI, MboI, and SspI. The RFLP profiles of all samples on digestion with the MboI enzyme yielded a fragment size of 229 and 362 bp except for vaccine strain Bursine Plus, which yielded a profile of 229 and 480 bp. However, digestion with BstNI yielded two distinct RFLP patterns. The first profile was detected in field isolates ML-1/SPVC/2001 and NP2/SPVC/2002 with four fragments of 119, 154, 172, and 209 bp, resembling RFLP profiles of molecular group 4 isolates. NL-3/SPVC/2003, NK-4/SPVC/2004, and NPK-5/SPVC/2005 generated a different RFLP profile with fragments of 119, 172, and 424 bp, resembling the profiles of molecular group 6 isolates. However, all the field and vaccine strains showed the absence of SspI restriction sites in their genome. It can be concluded that the Pakistani isolates can be grouped in molecular groups 4 and 6 of IBDV.